Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is the virus responsible for causing coronavirus disease 2019 (COVID-19), which was declared a pandemic by the World Health Organization (WHO) in March 2020 .
As of April 2022, the global number of infections was estimated at 500 million and a total of over 6.1 million deaths associated with COVID-19 have been recorded.
Although effective COVID-19 vaccines have been rapidly produced and implemented, the rate of new variants has increased the demand for vaccine formulation updates.
The production of substantial quantities of stable, high-quality SARS-CoV-2 S proteins is essential for the development of virosomal-based vaccines. Full-length protein S production has been reported using a variety of expression systems, most of which are based on mammalian cells. The insect cell-baculovirus expression vector system (IC-BEVS) is a viable option as it is widely considered a scalable and low-cost manufacturing platform.
In a recent study published in Pharmacydifferent signal peptides, baculovirus transfer vectors, cell lines, infection techniques and formulation buffers were studied with the aim of constructing a scalable bioprocess to generate high-quality protein S to be incorporated into a COVID-19 vaccine candidate at virosome basis.
The stability, oligomeric state, and ability to bind the generated protein to angiotensin-converting enzyme 2 (ACE2) receptor and selected neutralizing SARS-CoV-2 antibodies were all thoroughly assessed. Protein S was also covalently bound to a click chemistry lipid in the virosomal membrane through its polyhistidine (His) tag.
The most suitable method of infection was identified via the infection of sf-9 cells at a cell concentration at infection (CCI) of 1 and 2 x 106 cell/mL with rBac recombinant baculovirus with a multiplicity of infection (MOI) of 0.1 and 1 pfu/cell, and small scale (SF) shake flasks were used to examine the growth and kinetics of protein S expression. Following infection, traditional patterns of insect cell viability and growth were observed. CCI = 2 x 106 cell/mL and MOI = 1 pfu/cell produced the highest protein S titers and specific production rates.
The authors explored three different signal peptides, which included honey bee melittin (BVM) (rBac 1), rBac gp67 (rBac 2), and the S protein S signal peptide from the SARS-CoV-2 strain of origin (rBac 3). Sf-9 insect cells were infected at CCI = 2 × 106 cell/mL with each rBac at MOI = 1 pfu/cell, and small-scale SF was used to examine protein S expression and growth kinetics.
Following infection, the authors found traditional patterns of insect cell viability and growth, with rBac 1-infected samples being the only ones to have protein S detected by Western blot, therefore, baculovirus constructs containing the BVM signal sequence were used in future analyses.
For all N-linked glycan sites already identified in current literature, purified protein S was analyzed by liquid chromatography-mass spectrometry (LC-MS) to determine site-specific glycosylation and glycan composition. At glycosylation sites N68_81, N172, N241, and N1081, a combination of mannose-rich and complex/paucimannose-like glycans was discovered; the remaining 15 sites were dominated by complex-type transformed glycans.
High-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) and differential scanning fluorimetry (DFS) were used to examine the medium-term storage durability of isolated protein S. When stored at 80°C and 4°C or after 5 freeze-thaw cycles, HPLC-SEC analysis showed a single peak in all conditions tested, implying that the trimer structure of the protein S is maintained for up to 90 days. The durability of protein S was supported by DSF data, which revealed a minor difference in protein S melting temperatures under all circumstances studied.
Dibenzocyclooctyne-(DBCO-)azide click chemistry was used to covalently link virosomes to purified protein S, and an enzyme immunoassay (ELISA) was used to detect protein S on virosomes through exposed epitopes and ACE2 binding. The S protein outside the virosomes has the ability to bind to the ACE2 receptor and is also recognized by CR3022 and all neutralizing antibodies tested against various epitope clusters, according to the results.
This research shows that an insect cell-baculovirus expression vector system can be used to create a high-quality SARS-CoV-2 S protein for implementation in a virosome-based COVID-19 vaccine candidate. . The authors claim that the bioprocess engineering approach used here enabled them to produce 4 mg/L of full-length protein S, which is the highest value obtained to date using insect cells.
Additionally, protein S produced from Sf-9 insect cells showed mammalian-like glycan processing and medium-term storage durability. Moreover, even after one month of storage at 4°C, protein S outside the virosomes had the ability to bind to the ACE2 receptor and was recognized by a wide range of neutralizing antibodies. Immunogenicity and safety-toxicology investigations in appropriate animal models should be conducted to verify that these particles are candidate vaccines against COVID-19.